A Dermatological View—In vitro Buffering Capacity of Human Skin Layers*

* Modified with permission from Y Zheng, Buffering capacity of human skin layers: In vitro, Skin Research and Technology (2011).¹¹

Normal stratum corneum (SC) is acidic, with typical pH ranges from 4 to 6, and while skin exposed to aqueous acid or alkaline solutions exhibits changes in pH, it may rapidly restore to the baseline values. This phenomena is called buffering capacity. Many factors contribute to skin’s buffering capacity including keratin, proteins, sweat, SC thickness, free amino acids and other water-soluble epidermis constituents.¹ Previous studies demonstrate that skin buffering capacity can be measured in vitro by applying several concentrations of hydrogen chloride (HCl) and sodium hydroxide (NaOH) on skin and evaluating the pH change pre- and post-dosing.² Here, the authors employed this technique to evaluate the buffering capacity of skin layers including intact SC, denuded SC and dermis skin samples.

Materials

Skin: Human skin was dermatomed to 500 μm thickness and frozen at -20°C. Defrosted skin samples were removed by 40 continuous tape strips. The epidermis samples were heat-separated for 30 sec in a 60°C water bath.³ Skin samples were acclimatized for at least 30 min prior to baseline measurements.

Irritants (model acid and base): NaOH, HCl solutions and water (HPLC grade) were prepared as previously described.²

pH measurement: A skin pH meter^a was used to perform pH measurements. The electrode was mounted directly on the skin surface to obtain recordings; the principle of this device and measuring method were previously published.^{4, 5}

TEWL measurement: An evaporation meter^b was utilized to measure TEWL as previous described.^{6, 7} TEWL readings are expressed in g/m² per hr.

Methods

Split skin samples, approximately 5 cm² in surface area, dermatomed to 500 μm thickness, were mounted on liquid scintillation vials filled with saline, 0.9% sodium chloride (NaCl), as receptor fluid. After initial preparations, samples were equilibrated for 30 min. Prior to dosing, the baseline pH and TEWL values were recorded. Each sample was dosed with 3.18 μL/cm² of corresponding acid and base. Skin washed with de-ionized water and untreated skin were used as blank and negative controls, respectively. Immediately after dosing, as well as at 10 min and 30 min intervals, pH measurements were performed. Thirty minutes later, skin surfaces were washed twice with 1 mL of de-ionized water for 10 sec. The pH levels of washed skin were taken immediately and at 10 min and 30 min intervals. At the end of measurements, skin was cut with a punch and weighed by equi-armbalance.

Results

pH changes: pH values significantly changed (p < 0.05) immediately following NaOH exposure. Higher concentrations induced the greatest pH changes. Within a short time after the base exposure, the dermis demonstrated the strongest buffering capacity. At 30 min, intact skin demonstrated the strongest buffering capacity, while the dermis was ranked weakest (p < 0.01). Following washing, the pH values of the dermis more closely followed the baseline value; pH = 6.0 at 0 min and 10 min post-washing, compared to other layers. At 30 min post-washing, the intact skin’s pH values declined faster than the other layers (see Figure 1). The ability of HCl to modify skin pH was found to be comparable with the ability of NaOH, considering similar concentrations (see Figure 2).

Rate of Buffering Capacities

Buffering capacity: Figure 3 presents the rate of buffering capacity of skin layers which are calculated based on Eq. 1.

The dermis presented the greatest buffering capacity immediately after being exposed to acid or base. With time, the pH of the dermis changed less, compared with the other layers. The buffering capacity was highest in intact SC 30 min after application.

TEWL: The intact skin presented the lowest TEWL values while the dermis had the highest TEWL values— p = 0.041, compared with intact skin; the denuded SC model presented in the mid-values; p = 0.002 compared with the intact skin.

Discussion

The acidic property of normal skin plays a crucial role in regulating bacterial skin flora, sensitizing skin to cleansing procedures, and maintaining barrier homeostasis.^{1, 8, 9} Alterations in skin pH present a degree of diseased skin, undergoing acute or chronic modifications.^{9, 10} The baseline pH value for skin layers was within the 4 to 6 ± 0.2 range.¹ Although human cadaver skin may maintain partial viability for several days,¹⁰ the alteration of human skin pH after death is not clear.

The correlation between human cadaver skin pH and in vivo measurements should be studied to provide better insights about the validity of in vitro skin pH investigations. Previously, skin pH and its buffering capacity were demonstrated to be measurable in vitro by applying 0.025, 0.05 and 0.1N of HCL and NaOH on human cadaver skin and detecting pH alterations post-dosing and -washing stages. This study demonstrated that pH values of different skin layers could be easily measured on human cadaver skin.

The results shown here reveal that the skin buffering capacity of different skin layers substantially differ from each other. This finding could be a step in understanding issues related to dermatopharmacology and dermatotoxicology. Further investigations may also clarify relative buffering capacity and percutaneous penetration. However, due to limitations of in vitro skin pH buffering capacity measurements, such as in vivo buffering mechanisms involving oxygen and carbon dioxide, and bioavailability considerations, the present technique cannot fully replace in vivo investigations. By investigating the correlations between the described study and in vivo measurements, the clinical implications of these investigations may be more fully understood.

The authors conceptualize the complexity of the interaction of base/acid on the skin layers to involve not only percutaneous penetration kinetic (PK) and aggression kinetic, but constant pH change—with measuring penetration. Insufficient data is provided to propose a PK/PH model but the present system could provide the basis for such. This information will be especially important for optimizing cosmetics and skin care formulations of relatively high and low pH levels. Furthermore, the industry awaits the development of reliable data of in vivo models.

Conclusion

This study investigated the in vitro buffering capacity of human skin layers after exposure with a topically applied model acid and base. NaOH and HCl solutions were applied to intact SC, denuded SC and dermis skin samples. Results indicated that immediately following acid or base exposure, the dermis had the highest buffering capacity while with time, the buffering capacity of intact SC predominates. These findings present the potential to better understand skin’s buffering capacity as it relates to dermatopharmacology and the dermatotoxicology of skin care foundations.

References

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